Chemical Constituents and Antimicrobial Activity of a Ganoderma lucidum (Curtis.) P. Karst. Aqueous Ammonia Extract

Mushroom extracts have shown potential as a source of new antimicrobial agents. This study investigates the chemical profile of an aqueous ammonia extract obtained from the carpophores of Ganoderma lucidum, which grows on Quercus ilex trees, and explores its valorization as a biorational. The major chemical constituents of the extract, identified through gas chromatography–mass spectrometry, include acetamide, oleic acid, 1,2,3,4-butanetetrol, monomethyl azelate, undecane, and palmitic acid. The anti-oomycete and antifungal activity of G. lucidum extract was evaluated against Phytophthora cinnamomi, the primary threat to Quercus spp. in the dehesa biome, as well as three Botryosphaeriaceae fungi. In vitro tests revealed minimum inhibitory concentration (MIC) values of 187.5 μg·mL−1 against P. cinnamomi and 187.5–1000 μg·mL−1 against the fungi. Furthermore, conjugation of the G. lucidum extract with chitosan oligomers (COS) synergistically enhanced its antimicrobial activity, resulting in MIC values of 78.12 and 375–500 μg·mL−1 against P. cinnamomi and the fungi, respectively. These MIC values are among the highest reported to date for natural products against these phytopathogens. Subsequent ex situ testing of the COS-G. lucidum conjugate complex on artificially inoculated Q. ilex excised stems resulted in high protection against P. cinnamomi at a dose of 782 µg·mL−1. These findings support the potential utilization of this resource from the dehesa ecosystem to protect the holm oak, aligning with sustainable and circular economy approaches.


Introduction
Medicinal mushrooms' fruiting bodies, mycelium, and spores are valuable sources of bioactive products [1]. Ganoderma lucidum (Curtis.) P. Karst. is a dark, large fungus with a glossy exterior and a woody texture. It has been used for promoting health and longevity in Japan and China, where it is known as 'reishi' or 'mannentake', and 'lingzhi', respectively. The G. lucidum fruiting body has a tawny-to-russet-colored stipe ( Figure 1). The context tissue, cinnamon-buff to pink-buff in color, shows concentric growth zones.
The antimicrobial activity of G. lucidum extracts has been evaluated against bacteria such as Bacillus subtilis, Staphylococcus aureus, Klebsiella aerogenes, Corynebacterium diphtheriae, Escherichia coli, Salmonella spp., and Pseudomonas aeruginosa [2], as well as against fungi such as Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, Mucor indicus, Curvularia lunata, Fusarium oxysporum, Alternaria alternata, Drashelaria spp., and Penicillium spp. [3]. Yang et al. [14] demonstrated that G. lucidum polysaccharides combined with small amounts of chemical fungicides were successful in controlling plant diseases such as wheat brood, root rot, and corn stalk rot.
Concerning phytopathogens, Phytophthora spp. are a threat to global food security and the health, function, and biodiversity of native ecosystems [15]. The dehesa (semi-natural open woodlands) is a characteristic ecosystem of the Iberian Peninsula that is affected by one of these pathogens. The loss of trees due to the disease caused by the oomycete Phytophthora cinnamomi is one of the most significant problems that this biome faces, which is exacerbated by climate change [16]. Phytophthora cinnamomi is a globally distributed pathogen that can infect thousands of species and is considered to be the main biotic driver of Quercus spp. woodlands' decline in Spain [17]. It is also one of the most threatening invasive pathogens in the world [18]. In addition to P. cinnamomi, the trees in the dehesa are also threatened by ascomycete fungi of the genus Botryosphaeria, including Botryosphaeria dothidea, Diplodia corticola, and Dothiorella iberica. These fungi cause cankers and dieback of twigs and have been associated with the decay of holm oaks and cork oaks, although B. dothidea has also been found in other species of the genus Quercus such as Quercus robur L. and Quercus rubra Michx. L. [19].
Taking into consideration that the use of fungicides is discouraged under the current new European Union forest strategy for 2030 (Sustainable Forest Management in Europe, 2022/2016(INI)) and that Article 14 of Directive 2009/128/EC promotes the use of formulations based on natural ingredients as new protection techniques, the study presented herein aims to study the chemical constituents present in G. lucidum aqueous ammonia The biological activity of G. lucidum has been investigated by Mizuno et al. [7] and Liu et al. [8]. Its polysaccharide composition significantly contributes to G. lucidum's immunomodulatory, antioxidant, antitumor, and antibacterial properties [5,9]. On the other hand, its triterpene content is responsible for its antitumor, anti-inflammatory, antioxidant, anti-hepatitis, antimalarial, hypoglycemic, antimicrobial, and anti-inflammatory activity [10,11]. Furthermore, its polyphenol content plays a role in its antioxidant, antimicrobial, and anti-inflammatory properties, as well as its anti-tyrosinase activity [12,13].
The antimicrobial activity of G. lucidum extracts has been evaluated against bacteria such as Bacillus subtilis, Staphylococcus aureus, Klebsiella aerogenes, Corynebacterium diphtheriae, Escherichia coli, Salmonella spp., and Pseudomonas aeruginosa [2], as well as against fungi such as Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, Mucor indicus, Curvularia lunata, Fusarium oxysporum, Alternaria alternata, Drashelaria spp., and Penicillium spp. [3]. Yang et al. [14] demonstrated that G. lucidum polysaccharides combined with small amounts of chemical fungicides were successful in controlling plant diseases such as wheat brood, root rot, and corn stalk rot.
Concerning phytopathogens, Phytophthora spp. are a threat to global food security and the health, function, and biodiversity of native ecosystems [15]. The dehesa (semi-natural open woodlands) is a characteristic ecosystem of the Iberian Peninsula that is affected by one of these pathogens. The loss of trees due to the disease caused by the oomycete Phytophthora cinnamomi is one of the most significant problems that this biome faces, which is exacerbated by climate change [16]. Phytophthora cinnamomi is a globally distributed pathogen that can infect thousands of species and is considered to be the main biotic driver of Quercus spp. woodlands' decline in Spain [17]. It is also one of the most threatening invasive pathogens in the world [18]. In addition to P. cinnamomi, the trees in the dehesa are also threatened by ascomycete fungi of the genus Botryosphaeria, including Botryosphaeria dothidea, Diplodia corticola, and Dothiorella iberica. These fungi cause cankers and dieback of twigs and have been associated with the decay of holm oaks and cork oaks, although B. dothidea has also been found in other species of the genus Quercus such as Quercus robur L. and Quercus rubra Michx. L. [19].
Taking into consideration that the use of fungicides is discouraged under the current new European Union forest strategy for 2030 (Sustainable Forest Management in Europe, 2022/2016(INI)) and that Article 14 of Directive 2009/128/EC promotes the use of formulations based on natural ingredients as new protection techniques, the study presented herein aims to study the chemical constituents present in G. lucidum aqueous ammonia extract by gas chromatography-mass spectrometry (GC-MS) and to explore opportunities for the valorization of this extract for the control of aforementioned phytopathogens. This second

Antifungal and Anti-Oomycete Activity
The results of the antifungal/anti-oomycete susceptibility test are presented in Figure 3. An increase in concentration led to a decrease in the radial growth of the mycelium for all three tested products (COS, G. lucidum carpophore aqueous ammonia extract, and their conjugate complex), resulting in statistically significant differences. The aqueous ammonia extract of G. lucidum carpophores exhibited higher antifungal/anti-oomycete activity than COS, with minimum inhibitory concentrations (MICs) ranging from 187.5 to 1000 µg·mL −1 and from 750 to 1500 µg·mL −1 , respectively. Phytophthora cinnamomi was the most sensitive phytopathogen in both cases, with MIC values of 187.5 and 750 µg·mL −1 for G. lucidum extract and COS, respectively. The formation of conjugate complexes improved the activity, with the COS-G. lucidum conjugate producing complete inhibition of Botryosphaeriaceae family pathogens at concentrations in the range of 375 to 500 µg·mL −1 , while the inhibition value was as low as 78.12 µg·mL −1 for P. cinnamomi. The 50 and 90% effective concentrations (EC 50 and EC 90 , respectively), presented in Table 3, allow for a clearer observation of this enhancement of the antifungal/anti-oomycete activity, which was quantified according to Wadley's method. The synergy factor values were in the range of 1.98-3.63. As these values were higher than 1, a synergistic behavior can be inferred in all cases.

Antifungal and Anti-Oomycete Activity
The results of the antifungal/anti-oomycete susceptibility test are presented in Figure  3. An increase in concentration led to a decrease in the radial growth of the mycelium for all three tested products (COS, G. lucidum carpophore aqueous ammonia extract, and their conjugate complex), resulting in statistically significant differences. The aqueous ammonia extract of G. lucidum carpophores exhibited higher antifungal/anti-oomycete activity than COS, with minimum inhibitory concentrations (MICs) ranging from 187.5 to 1000 µg·mL −1 and from 750 to 1500 µg·mL −1 , respectively. Phytophthora cinnamomi was the most sensitive phytopathogen in both cases, with MIC values of 187.5 and 750 µg·mL −1 for G. lucidum extract and COS, respectively. The formation of conjugate complexes improved the activity, with the COS-G. lucidum conjugate producing complete inhibition of Botryosphaeriaceae family pathogens at concentrations in the range of 375 to 500 µg·mL −1 , while the inhibition value was as low as 78.12 µg·mL −1 for P. cinnamomi. The 50 and 90% effective concentrations (EC50 and EC90, respectively), presented in Table 3, allow for a clearer observation of this enhancement of the antifungal/anti-oomycete activity, which was quantified according to Wadley's method. The synergy factor values were in the range of 1.98-3.63. As these values were higher than 1, a synergistic behavior can be inferred in all cases.  For the purpose of comparison, Fosetyl-Al, a conventional synthetic fungicide widely employed against Phytophthora spp. and fungi associated with grapevine trunk diseases (GTDs), was utilized as a positive control. As indicated in Table 4, when administered at the recommended dose of 2000 µg·mL −1 (equivalent to 2.5 g·L −1 for Fosbel ® , fosetyl-Al 80%), complete inhibition of the four phytopathogens was observed. However, when applied at one-tenth of the recommended dose, a moderate inhibition was observed against B. dothidea and D. corticola, while a weak inhibition was observed in the case of P. cinnamomi, and no inhibition was detected against D. iberica. The radial growth of the mycelium for the control (PDA only) was 75 mm. All mycelial growth values (in mm) are average values (n = 3).

Protection of Excised Stems against P. cinnamomi
The COS−G. lucidum conjugate complex was the most active product in the in vitro tests and was subsequently tested as a protective treatment against P. cinnamomi on holmoak-excised stems. Three different concentrations were used, corresponding to the MIC, MIC × 5, and MIC × 10 (i.e., 78, 391, and 782 µg·mL −1 , respectively). Results are presented in Figure S2, and a comparison of canker lengths is shown in Table 5. No protective effect was observed at the lowest dose tested (i.e., at the MIC value obtained in the in vitro tests), with canker lengths similar to those of the positive control (non-treated stems infected with the oomycete). At a dose equal to five times the MIC, significantly lower canker lengths were observed. However, it was necessary to increase the concentration up to 10 times the MIC to achieve effective protection, with no significant differences compared to the negative control. Nevertheless, at this dose, small cankers were still visible in four of the excised stems (out of fifteen replicates), indicating that higher doses may be required in field conditions.

On the Chemical Profile
Among the list of compounds presented in Table 2, acetamide or ethanamide has been previously identified in red beetroots (Beta vulgaris var. rubra) and Clerodendrum infortunatum L. leaves [20]. It has also been found in extracts from Larrea divaricata Cav., Picea pungens Engelm., and Sequoiadendron giganteum (Lindl.) Buchholz. The presence of acetamide in the extract may be attributed to the partial hydrolysis of N,N-dimethylacetoacetamide, which was also identified in the extract. Alternatively, it could originate from N-(3methylbutyl)acetamide or N(2-phenylethyl)acetamide, which are common components of fresh wild mushrooms [21]. However, it is worth noting that the presence of acetamide in the extract may be an artifact resulting from the extraction procedure, as it can also be formed through the decomposition of ammonium acetate. Ammonium acetate is generated by neutralizing excess ammonia in the extract with acetic acid. It is important to mention that acetamide-containing compounds are widely used as herbicides in agriculture [22], and several acetamide derivatives have been reported to act as antimicrobial agents [23].
L-threitol, also known as 1,2,3,4-butanetetrol, is a non-cariogenic component found in Shiitake mushrooms [34] and is also the primary component of Thaumatococcus daniellii (Benn.) Benth. ex B.D.Jacks. leaves [35]. At present, there is no available information on the antimicrobial, antibacterial, or antifungal activity of this compound.
Nonanedioic acid (or 8-carbomethoxyoctanoic acid) monomethyl ester, also known as monomethyl azelate, is a dicarboxylic acid naturally produced by Malassezia furfur (C.P. Robin) Baill. and is also present in whole-grain cereals, rye, and barley. It is known to be effective in treating acne and various cutaneous disorders [36].

On the Antimicrobial Activity Comparison of G. lucidum Extracts
The antibacterial and antifungal activity results reported for G. lucidum aqueous ammonia extract in this study are consistent with the previously reported antimicrobial activity of G. lucidum extracts in other solvents (Table S1) [3,12,[53][54][55][56][57][58][59]. However, previous reports have primarily focused on human pathogens, with limited data on phytopathogens, thus making a direct comparison among extraction media unfeasible.

Comparison of Efficacy vs. Other Natural Compounds
The use of different isolates with distinct susceptibility profiles generally makes it difficult to accurately compare the activity of G. lucidum aqueous ammonia extract with that of other plant extracts reported in the literature (see Table 6). Nevertheless, it can be observed that G. lucidum-based treatments exhibit some of the highest activities against the four phytopathogens. Regarding B. dothidea, the efficacy of the pure extract is comparable to that of a compound herbal extract compound consisting of seven Chinese medicinal plants [60]. Meanwhile, the activity of the conjugate complex is intermediate between those of COS-U. dioica and COS-E. arvense conjugates [41], tested against the same isolate. Concerning D. corticola, the extract displays the highest activity. As for D. iberica, the data are only available for COS-U. dioica and COS-E. arvense conjugates [41] (tested against the same isolate), which exhibited lower activity, with MIC values at least twice that of the COS-G. lucidum conjugate complex. In terms of the activity against P. cinnamomi (MIC = 187.5 for G. lucidum extract), it is only lower than those reported for an aqueous ammonia extract of holm oak bark (MIC = 78.12 µg·mL −1 ) [61] and O. ficus-indica aqueous extract (EC 90 = 121.7 µg·mL −1 ) [62], and comparable to those of Flourensia cernua DC. extract (EC 90 = 193.4 µg·mL −1 ) [62] and Thymus vulgaris L. essential oil (MIC = 200 µg·mL −1 ) [63].

Comparison of Efficacy vs. Fosetyl-Al
Upon comparing the values of mycelial growth inhibition for Fosetyl-Al (as shown in Table 4) with the effective concentrations reported for G. lucidum extract and its conjugate complexes (as presented in Table 3), it can be observed that the in vitro activity of the natural products was comparable to or even higher than that of the conventional fungicide. Specifically, in the case of P. cinnamomi, complete inhibition was achieved at concentrations of 187.5 µg·mL −1 and 78.1 µg·mL −1 for the non-conjugated extract and the conjugate complex with COS, respectively, whereas Fosetyl-Al exhibited only 12% inhibition at a concentration of 200 µg·mL −1 .

Comparison of Efficacy in Excised Stems
Concerning the activity of the COS-G. lucidum extract conjugate complex as a protective treatment against P. cinnamomi, a comparison with other treatments against Phytophthora spp. is presented in Table 7. Its efficacy was similar to that of non-conjugated Q. ilex aqueous ammonia extract [61], although it was tested on Prunus amygdalus × P. persica excised stems rather than on Q. ilex ones. The activity of the COS-G. lucidum extract conjugate complex was higher than those of non-conjugated Sambucus nigra L. flower ammonia extract [73] and the COS-Quercus suber L. aqueous ammonia bark extract conjugate complex [74], but these were tested against Phytophthora cactorum and Phytophthora megasperma, respectively, so the comparison should be made with caution.

Reagents and Fungal Isolates
Ammonium hydroxide (50% v/v aqueous solution) was purchased from Alfa Aesar Specimens were identified and authenticated by Prof. Dr. B. Herrero-Villacorta (Departamento de Ciencias Agroforestales, ETSIIAA, Universidad de Valladolid) and voucher specimens are available at the herbarium of the ETSIIAA. Different specimens (n = 20) were thoroughly mixed to obtain composite samples, which were shade-dried, pulverized to a fine powder in a mill grinder, homogenized, and sieved (1 mm mesh).

Extraction Process, Preparation of Chitosan Oligomers, and Preparation of Conjugate Complexes
An aqueous ammonia extraction medium was chosen due to the woody texture of G. lucidum and to achieve the dissolution of polyphenols and other bioactive compounds of interest. Briefly, 67.3 g of G. lucidum carpophore powder was first digested in an aqueous ammonia solution (140 mL H 2 O + 20 mL NH 3 ) for 2 h, then sonicated in pulsed mode (with a 2 min stop every 2.5 min) for 10 min using a probe-type ultrasonicator (model UIP1000hdT; 1000 W, 20 kHz; Hielscher Ultrasonics, Teltow, Germany), and then allowed to stand for 24 h. It was neutralized to pH 7 using acetic acid. Finally, the solution was centrifuged at 9000 rpm for 15 min, and the supernatant was filtered through Whatman No. 1 paper. The extraction yield was 4.2% (2.86 g).
Aliquots of the extract were freeze-dried for attenuated total-reflectance Fouriertransform infrared (ATR-FTIR) spectroscopy and GC−MS characterization. For the latter, 25 mg of the lyophilized extract was resuspended in 5 mL of methanol (HPLC grade) to obtain a 5 mg·mL −1 solution, which was filtered before injection.
Chitosan oligomers were prepared using the method previously reported in [75], resulting in a solution with oligomers with a molecular weight of less than 2 kDa.
The COS-G. lucidum carpophore extract conjugate complex was obtained by combining solutions (both at a concentration of 3000 µg·mL −1 ) in a 1:1 (v/v) ratio, followed by sonication for 15 min (five pulses lasting 3 min each to keep the temperature below 60 • C). The solution was freeze-dried for ATR-FTIR characterization to confirm the formation of the conjugate complex.

G. lucidum Characterization Procedures
The infrared vibrational spectra of the G. lucidum dried samples, as well as that of a commercial G. lucidum sample, were registered using a Thermo Scientific (Waltham, MA, USA) Nicolet iS50 FTIR spectrometer, equipped with an in-built diamond ATR system. The spectra were collected over the 400-4000 cm −1 range, with a 1 cm −1 spectral resolution, taking the interferograms resulting from co-adding 64 scans.
The aqueous ammonia extract of G. lucidum carpophores was analyzed by GC-MS at the Research Support Services (STI) at Universidad de Alicante (Alicante, Spain), using an Agilent Technologies gas chromatograph model 7890A coupled to a quadrupole mass spectrometer model 5975C. The chromatographic conditions were as follows: injection volume = 1 µL; injector temperature = 280 • C, in splitless mode; initial oven temperature = 60 • C, 2 min, followed by a ramp of 10 • C/min up to a final temperature of 300 • C, 15 min. The chromatographic column used for the separation of the compounds was an Agilent Technologies HP-5MS UI column with a length of 30 m, a diameter of 0.250 mm, and a film thickness of 0.25 µm. The mass spectrometer conditions were as follows: temperature of the electron impact source of the mass spectrometer = 230 • C; temperature of the quadrupole = 150 • C; ionization energy = 70 eV. The identification of components was based on a comparison of their mass spectra and retention time with those of the authentic compounds and by computer matching with the database of the National Institute of Standards and Technology (NIST11).

In Vitro Antifungal and Anti-Oomycete Activity
The antifungal and anti-oomycete activity of the G. lucidum carpophore extract and the conjugate complex with COS was examined using the poisoned food method. Aliquots of stock solutions were added to the PDA medium to produce final concentrations in the range of 15.62-1500 µg·mL −1 . Mycelial plugs were transferred from the margin of one-week-old PDA cultures of B. dothidea, D. corticola, D. iberica, and P. cinnamomi to plates filled with the amended media. For each treatment and concentration combination, three plates were used, and each experiment was carried out twice. The untreated control consisted of replacing the extract with the solvent used for extraction in the PDA medium. Fosbel ® (fosetyl-Al 80%, reg. no. 25502; Probelte, Murcia, Spain) was used as a positive control. Additional controls, consisting of pure PDA medium and PDA with the lowest concentration of the treatment, were also included to confirm the absence of contamination. Radial mycelium growth was quantified by measuring the average of two perpendicular colony diameters for each replicate. Growth inhibition was estimated after incubation in the dark at 25 • C for one week, using the formula: ((d c − d t )/d c ) × 100, where d c is the average colony diameter in the untreated control and d t is the average diameter of the treated colony. Effective concentrations (EC 50 and EC 90 ) were estimated using PROBIT analysis in IBM SPSS Statistics v.25 (IBM; Armonk, NY, USA). The degree of interaction was estimated using Wadley's method [76].

Protection Tests on Artificially Inoculated Excised Stems
Given the restrictions that apply to in vivo assays involving P. cinnamomi, the efficacy of the most active treatment in the in vitro tests (i.e., COS−G. lucidum carpophore extract conjugate complex) was investigated by artificial inoculation of excised stems in controlled laboratory conditions. Inoculation was performed according to the procedure proposed by Matheron et al. [77], with modifications as described in [61,73,74]. Young stems (1.5 cm diameter) of healthy Q. ilex plants were cut into 10 cm-long sections using a sterilized grafting knife. The excised stem pieces were immediately wrapped in moistened sterile absorbent paper. In the laboratory, the freshly excised stem segments were first immersed in a 3% NaClO solution for 10 min, then in 70% ethanol for 10 min, and then thoroughly rinsed four times with distilled water, to avoid superficial contaminants in the tissue. Some of the stem segments (n = 15 for the positive control, and n = 15 for the negative control) were soaked for 1 h in distilled water to be used as controls, while the remaining stem segments were soaked for 1 h in aqueous solutions containing an appropriate amount of the conjugate complex to obtain MIC, MIC × 5, and MIC × 10 concentrations (n = 15 segments/concentration). A coadjuvant (Alkir ® , 1% v/v) was added to all the solutions, including the control, to facilitate the moistening and penetration of the treatment into the bark. After soaking, the stem pieces were allowed to dry, and the bark was carefully removed with a scalpel to reveal the cambium. The bark was then placed on an agar Petri dish and, in the case of the positive control and treated samples, it was inoculated by placing a plug (diameter = 5 mm) from the margin of a one-week-old PDA culture of P. cinnamomi on the center of the inner surface of the bark. After inoculation, stem segments were incubated in a humid chamber for 4 days at 24 • C and 95-98% relative humidity. The efficacy of the treatments was evaluated by measuring the lengths of the cankers that developed at the inoculation sites. Finally, the oomycete was re-isolated from the lesions and morphologically identified to fulfill Koch's postulates.

Statistical Analysis
The results from the in vitro mycelial growth inhibition and ex situ necrosis lengths were subjected to statistical analysis using one-way analysis of variance (ANOVA). Post hoc comparisons of means were conducted using Tukey's test at a significance level of p < 0.05. Homogeneity and homoscedasticity requirements were checked using Shapiro-Wilk and Levene tests. The statistical analysis was performed using IBM SPSS Statistics v.25 software.

Conclusions
This study provides valuable insights into the composition and antimicrobial activity of an aqueous ammonia extract of Ganoderma lucidum carpophores. The GC-MS characterization revealed the presence of chemical constituents such as oleic acid and its methyl ester, 1,2,3,4-butanetetrol, monomethyl azelate, undecane, and palmitic acid and its methyl ester, which have demonstrated antimicrobial properties in previous studies. In vitro tests demonstrated significant anti-oomycete and antifungal activity of the G. lucidum extract, further enhanced upon combination with chitosan oligomers. In particular, conjugate complexes based on the extract exhibited notable efficacy against Phytophthora cinnamomi, a serious threat to Quercus spp., resulting in complete inhibition at 78.12 µg·mL −1 , which was confirmed in ex situ bioassays on holm-oak-excised stems. These findings highlight the potential of G. lucidum as a natural alternative to synthetic fungicides for controlling plant diseases caused by oomycetes and fungi, and suggest its promise as a bioactive product for safeguarding Quercus spp. in the dehesa ecosystem.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.